This application proposes a novel approach to improving the safety of opioid analgesics by post-receptor pharmacological targeting of G protein ?? subunits to modify the actions of ?-opioid receptors (MORs). Our laboratory has identified small molecule inhibitors of G protein ?? subunits that demonstrate in vivo efficacy in various animal models of disease. Relevant to this application, we identified two related G?? inhibitors, M119 and gallein that increase MOR agonist analgesic potency in mice without potentiating side effects that include development of tolerance, respiratory depression, constipation or addiction. Thus co-administration of G?? inhibitors with opioid analgesics has the potential to improve their safety profile by opening up the therapeutic window between analgesic efficacy and deleterious side effects. We propose that gallein and M119 modify opioid action by blocking specific feedback pathways downstream of MORs while leaving pro-analgesic pathways intact. MORs couple to the Gi family of G proteins and promote analgesia through G protein-dependent inhibition of neurotransmitter release via G??-dependent regulation of K+ (GIRK) channels, N-type Ca2+ channels and inhibition of vesicle fusion with synaptic membranes. At the same time G?? activates feedback pathways including phospholipase C (PLC?) and G protein-coupled receptor kinases (GRKs), which limit opioid receptor activity. Gallein and M119 block G??-dependent regulation of PLC? and GRK2 without blocking GIRK or N-type Ca2+ channels and thereby biasing MORs toward pro-analgesic signaling. Gallein and M119 have been powerful probe compounds to validate the idea that pharmacological G?? blockade could improve the properties of opioid analgesics, but the chemical characteristics of the molecules makes them unsuitable for therapeutic development. Data with new chemical series? derived from our high throughput screening (HTS) campaign support a G?? on-target mechanism of action. The primary goal of this application is to develop and mechanistically characterize novel ?drug like? G?? inhibitors that can be utilized to improve the safety of opioid analgesics. This application is divided into 3 specific aims 1) We will diversify the chemistry of promising lead compounds derived from HTS with the goal of improving potency and ?drug like? characteristics. We expect that upon completion of this aim we will identify a high potency ?drug like? G?? inhibitor that can be a strong lead candidate for therapeutic development. 2) We will use whole animal PLC?? and ?-arrestin-2 knockout models, and brain slice electrophysiology to examine the roles of blocking feedback inhibition of MOR signaling by PLC, PKC and ?-arrestin pathways in the potentiating actions of G?? inhibitors. 3) There is a significant need to find an opioid analgesic for the treatment of chronic pain without abuse potential and adverse side effects. We will explore the utility of G?? inhibition in chronic opioid use in a mouse model of chronic inflammatory pain, with both chronic and acute administration and use a rat model of morphine self-administration.